Table of Contents

• Coverage
• Dataset Description
  • Methods & Sampling
  • Data Processing Description
• Data Files
• Supplemental Files
• Related Publications
• Parameters
• Instruments
• Project Information
• Funding

Cell-cell interaction systems: the device utilized for the indirect co-culture system was Corning Transwell inserts (Corning Incorporated Life Science, Tewsbury, MA, USA) in the format of a 6-well plate with a permeable membrane (pore size: 0.4 µm). Gridded glass coverslips (ibidi USA Incorporation, Fitchburg, WI, USA) were used for cell attachment in the direct co-culture system.

The materials used to fabricate the single-probe include dual-bore quartz tubing (O.D. 500 µm, I.D. 127 µm, Friedrich & Dimmock, Inc., Millville, NJ, USA) and fused silica capillary (O.D. 105 µm, I.D. 40 µm, Polymicro Technologies, Phoenix, AZ, USA). The Single-probe was fabricated following our published protocols. Briefly, three major components (i.e., a Nano-ESI emitter, a dual-bore quartz tip, and a fused silica capillary) were integrated to prepare a Single-probe. Dual-bore quartz needles were produced by pulling the dual-bore quartz tubing using a laser micropipette puller (Sutter P-2000, Sutter Instrument, Novato, CA). The nano-ESI emitters were pulled from the fused silica capillaries using a butane micro torch. A Single-probe was fabricated by embedding a fused silica capillary and a nano-ESI emitter into those two channels of a dual-bore quartz needle. The Single-probe was coupled to a Thermo LTQ Orbitrap XL mass spectrometer for SCMS analysis. The sampling solvent (acetonitrile supplemented with 1% formic acid) was used for the SCMS experiment at a flow rate of ~0.05 µl/min.

Instruments
The Thermo LTQ Orbitrap XL mass spectrometer (Thermo Scientific, Waltham, MA, United States) was used. The SCMS experiment parameters included a mass range of m/z 200-1500 for positive ion mode and m/z 50-900 for negative ion mode, mass resolution 60,000, ionization voltage 4.5 kV, 1 microscan, and 100 ms max injection time.

Data processing
Single cell data was exported from the mass spec .raw files into Excel Sheets and submitted to BCO-DMO.   Each individual table was concatenated into a cohesive table (see BCO-DMO data manager processing notes).

Metabolites data from single cells were searched against three online metabolomics databases (Metlin, HMDB, and GNPS) for tentative labeling. Tandem MS (MS2) analyses were performed for molecular structure verifications.

BCO-DMO Data Manager Processing Notes:
* All Sheets from Excel files "Cell-cell interaction project_neg.xlsx" and "Cell-cell interaction project_pos.xlsx" were exported as a csv file per sheet. These sheets were bundled together with a sheet inventory providing experimental metadata to and made available as a supplemental file "single_cell_csvs.zip." 
* Each cell table (csv) was imported into the BCO-DMO  data system totaling 199 tables.  These were concatenated into a cohesive table with additional columns to for metadata contained in the original excel filename,  and sheet name.
* Additional Columns "Ion_mode" "Cell_Line" "Cell_Number" and "Description" were added to the combined data table using a lookup table provided by the data submitter that matched information about each Sheet position in the Excel file and which Cell line and cell number it corresponded to.
* sheet inventory and combined data table were reviewed by the original data submitter to ensure the provided metadata was represented accurately in the published data.

NSF Award Abstract:
High rates of dissolved organic nitrogen (DON) production and utilization in aquatic systems are typically attributed to microbial activity. Though it is known that there is a tight coupling between the production and consumption of biologically available DON, the composition, dynamics, and ecological significance of this rapidly cycled DON pool are less well understood. This proposal focuses on a component of the DON pool, creatine, which is historically understood as a product of metazoan activity, but appears to be both produced by phytoplankton and consumed by marine bacteria. Creatine is present in seawater in measurable quantities, which led to the hypothesis that creatine may be a significant component of the marine DON cycle. DON cycling likely has a bearing on fundamental marine ecosystem processes with large implications for carbon and nitrogen turnover on a global scale. Broader impacts of this project will include outreach that focuses on connecting scientists with K-12 students through research experiences for teachers and lesson development in collaboration with the K20 Center for Educational and Community Renewal, a statewide education research and development center at the University of Oklahoma. The project will integrate the research with inquiry-based teaching of rural secondary science teachers through Authentic Research Experiences in oceanographic science and microbial ecology. The K20 network includes 96% of Oklahoma schools, providing a unique opportunity to impact STEM education in Oklahoma.

The results of this project will help develop a better understanding of DON cycling, the ecological context of creatine uptake activity, and identify both creatine-producing and consuming organisms in the marine environment. The importance of creatine cycling will be assessed via 15N tracer studies along the natural coastal-to-offshore productivity gradient observed in the North Atlantic. Tracer and molecular approaches will be used to investigate the importance of phytoplankton vs. bacteria in creatine uptake and, the taxonomic identities of creatine-utilizing bacteria will be interrogated via molecular, stable isotope probing (SIP), and RT-qPCR approaches.